A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.
A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.
Prod'hom
G
G
Centre national de référence des mycobactéries, Institut Pasteur, Paris, France.
Lagier
B
B
Pelicic
V
V
Hance
A J
AJ
Gicquel
B
B
Guilhot
C
C
D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D016680:Genome, Bacterial; D016254:Mutagenesis, Insertional / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial